1. Field of the Invention
This invention relates to nucleic acid hybridization assay methods and reagent systems for detecting specific polynucleotide sequences. The principle of nucleic acid hybridization assays was developed by workers in the recombinant DNA field as a means for determining and isolating particular polynucleotide base sequences of interest. It was found that single stranded nucleic acids, e.g., DNA and RNA, such as obtained by denaturing their double stranded forms, will hybridize or recombine under appropriate conditions with complementary single stranded nucleic acids. By labeling such complementary probe nucleic acids with some readily detectable chemical group, it was then made possible to detect the presence of any polynucleotide sequence of interest in a test medium containing sample nucleic acids in single stranded form.
In addition to the recombinant DNA field, the analytical hybridization technique can be applied to the detection of polynucleotides of importance in the fields of human and veterinary medicine, agriculture, and food science, among others. In particular, the technique can be used to detect and identify etiological agents such as bacteria and viruses, to screen bacteria for antibiotic resistance, to aid in the diagnosis of genetic disorders such as sickle cell anemia and thalassemia, and to detect cancerous cells. A general review of the technique and its present and future significance is provided in Biotechnology (August 1983), pp. 471-478.
2. Description of the Prior Art
The state-of-the-art nucleic acid hybridization assay technqiues involve chemical modification of either the probe nucleic acid or sample nucleic acids for the purpose of labeling and detection. The necessity of chemically modifying nucleic acids severely limits the practical use of the technique since it requires the large-scale preparation of labeled probes involving complicated and expensive synthetic and purification procedures or the in situ synthesis of labeled sample nucleic acids by the analytical user. In particular, the resulting labeled polynucleotide must retain the ability to hybridize efficiently with its complementary sample or probe sequence. Such a requirement severely limits the availability of useful synthetic approaches to label modification of polynucleotides intended for use in hybridization assays.
The early hybridization techniques involved the use of radioactive labels such as .sup.3 H, .sup.32 P, and .sup.125 I. Labeled probes are synthesized enzymatically from radiolabeled nucleotides and a polynucleotide by such techniques as nick translation, end labeling, second strand synthesis, reverse transcription, and transcription. Thus, an additional requirement of such enzymatic methods is that the modified or labeled nucleotides must serve as effective substrates for the polymerase enzymes involved in the assembly of the labeled polynucleotide. Direct chemical modification of the polynucleotide is also possible, however, such a method is quite inefficient in incorporating labels into the polynucleotide and can affect the ability of the polynucleotide to undergo hybridization.
Because of the handling and storage disadvantages of radiolabeled materials, there has been considerable continuing efforts to develop useful nonradioisotopic labeling approaches. Such labels have included light emitting molecules such as fluorescers and chemiluminescers, and ligand molecules which are capable of being specifically bound by counterpart binders which are in turn labeled with detectable chemical groups such as fluorescers and enzymes. Examples of ligand labels are haptens, which are specifically bound by antibodies, and other small molecules for which specific binding proteins exist, e.g., biotin which is bound by avidin.
British Pat. No. 2,019,408 describes polynucleotide probes which are labeled with biotin through cytochrome C linking groups and which are then detectable by enzyme-labeled avidin. An alternative approach to labeling probes with low molecular weight ligands such as biotin is described in European Pat. Appln. No. 63,879. In this technique, 5-allylamine-deoxyuridine triphosphate (dUTP) derivatives are condensed with the desired ligand label and the thus modified nucleotide is incorporated by standard enzymatic methods into the desired probe. The use of light emitting labels is suggested by European Pat. Appln. Nos. 70,685 and 70,687. Other representatives of the patent literature pertaining to hybridization assays are U.S. Pat. Nos. 4,302,204 concerning the use of certain water soluble polysaccharides to accelerate hybridization on a solid-phase; 4,358,535 concerning the detection of pathogens in clinical samples; and 4,395,486 concerning the detection of sickle cell anemia trait using a synthetic oligonucleotide probe.
Techniques for detecting directly the polynucleotide duplex formed as the product of hybridization between the sample and probe polynucleotides, and thereby dispensing with the chemical labeling of one or the other polynucleotide, have been generally unsuccessful. Attempts to generate antibodies which will selectively bind double stranded DNA/DNA hybrids over single stranded DNA have failed [Parker and Halloran, "Nucleic Acids in Immunology", ed. Plescia and Braun, Springer-Verlag, NY(1969) pp. 18 et seq]. Some success has been achieved in generating antibodies that will bind RNA/DNA mixed hybrids and have low affinity for the single stranded polynucleotides [Rudkin and Stollar, Nature 265:472(1977); Stuart et al, PNAS(USA)78:3751(1981); Reddy and Sofer, Biochem. Biophys. Res. Commun. 103:959(1981); and Nakazato, Biochem. 19:2835(1980)], however, the sensitivity of these methods has not reached the levels required for clinical hybridization tests and one would have to use RNA probes which are well known to be quite unstable.
Accordingly, there is an established need for a technique for detecting hybridization without requiring chemical modification of polynucleotides or involving a labeling method of relative simplicity. Further, such technique should enable the use of a variety of labels, particularly of the nonradioisotopic type. A nucleic acid hybridization assay method and reagent system having these and other advantages are principal objectives of the present invention.
U.S. Pat. No. 4,257,774 describes a method for detecting various compounds that interact with nucleic acids, particularly compounds suspected as possible mutagens or carcinogens, by measuring the ability of such compounds to inhibit the binding of intercalators such as acridine orange to nucleic acids. Poirier, M.C. et al (1982) PNAS 79:6443-6447 describe the preparation of a monoclonal antibody selective for certain cis-platinum/double stranded DNA complexes over the free cis-platinum compound and double stranded DNA.